This project is focused on correlating the morphology of the node of Ranvier with its biochemistry and functionality. Particular emphasis will be on the differentiated membrane and cytoskeletal specialization of the nodal and paranodal regions. The data generated by this project is expected to contribute to an understanding of how the axon and the ensheathing cell interact, both with each other and with their local environment during development, degeneration, and regeneration, and during nerve conduction. The techniques that will be used are freeze- etching electron microscopy (EM), thin-section and thick section EM, selective staining, immunocytochemical labeling, immuno-EM localization, in situ hybridization, video-enhanced Nomarski light microscopy, laser confocal scanning microscopy, electrophysiology and the recording of signals from optical dyes. We will use the National High Voltage Electron Microscope (HVEM) Facility in Boulder, Colorado and the Regional Resource for Intermediate HVEM and Image Analysis in San Diego to obtain three-dimensional images from selectively stained structures or by serial section reconstruction. The following aims of the project are subdivided into areas of: structure; function; and development and recovery from demyelinative insult: 1) Structural Studies: Electron microscopy will be conducted on specimens prepared by rapid-freezing and deep-etching to complete our description of the cytoskeletal and extracellular matrix associations of the axonal and glial elements for both CNS and PNS nodes. In immunolabeling studies we will identify structures including: a) plasma membrane proteins such as Na channels, K channels, and the Na/K ATPase; b) internal membrane proteins like the ryanodine binding protein; c) cytoskeletal proteins like intermediate filaments, tubulins, spectrin, actin, myosin, alpha actinin, and ankyrin; d) extracellular matrix constituents like heparan sulfate proteoglycan, NCAM, and cytoactin. 2) Function: Our second aim is to correlate structural changes with physiological mechanisms of impulse conduction at the node. This is possible by in vitro electrophysiological recording of impulses at the nodes of Ranvier in PNS fibers that are simultaneously observed at high optical resolution using video-enhanced Nomarski differential interference contrast ((DIC) time-lapse recording in conjunction with optical methods for recording membrane potentials and Ca transients. This capability provides a unique opportunity to test hypotheses regarding ion channel locus, density and function of cell compartments at node of Ranvier. 3) Development and Remyelination: Our third aim is to determine the sequence of development of macromolecular components of the nodal complex with a special interest in determining if one of the constituents disclosed during the antibody localization studies conducted in aim (1) appears prior to the arrival of myelinating cells thus participating in the early definition of the node of Ranvier. We plan to establish a panel of antibody probes to probe the cellular mechanisms by which nodal sites are first defined during development as well as redefined in mature nervous systems following traumatic insults. An increase in our understanding of the dynamic cellular interactions that establish, maintain, and control axonal membrane protein complexes requisite for conduction of action potentials unquestionably will be of value in recognizing the effects of disease processes at these sites.